In eukaryotic cells, most transcripts synthesized by RNA polymerase II contain introns that are removed by splicing before transport to the cytoplasm. Unspliced mRNAs are usually excluded from the cytoplasm. Although the molecular basis for the nuclear retention of unspliced transcripts is generally unclear, it seems likely that either the process of splicing positively affects RNA transport, or the presence of spliceosomes on transcripts negatively affects RNA export from the nucleus. Based on experiments in a yeast system, it has been proposed that intron-containing mRNAs are retained in the nucleus due to association with spliceosomes (Legrain & Rosbash, Cell 57:573-583 (1989)). In mammalian systems, similar observations have been made. Chang and Sharp reported enhanced cytoplasmic accumulation of unspliced mRNAs when splice sites present in nascent transcripts were incapable of efficient spliceosome formation (Chang & Sharp, Cell 59:789-795 (1989)).
Splicing is not always a prerequisite for efficient cytoplasmic accumulation of mRNAs. In retroviruses, for example, alternative splicing of a single viral pre-mRNA generates multiple RNA products, a significant fraction of which are partially spliced or fully unspliced. These intron-containing mRNAs encode viral structural proteins, and their cytoplasmic accumulation is essential for the viral life cycle. In the case of HIV-1, efficient cytoplasmic accumulation of singly spliced and unspliced viral mRNAs requires a viral regulatory protein, called Rev, which interacts with the Rev-responsive element (RRE) present in the target transcripts (reviewed in Cullen, Microbiol. Rev. 56:375-394 (1992)). However, HIV RRE only works in some cell types; for example, it does not work in mouse cells.
For simple retroviruses, such as the Mason-Pfizer monkey virus, cytoplasmic localization of unspliced viral mRNAs involves the interaction of a cis-acting RNA element with an unidentified cellular factor(s) (Bray et al., Proc. Natl. Acad. Sci. USA 91:1256-1260 (1994)). Thus, specific interactions between positive cis-acting RNA elements and appropriate viral or cellular factors appear to facilitate the cytoplasmic accumulation of intron-containing retroviral mRNAs.
Some viral genes naturally lack introns. Examples include the hepatitis B virus (HBV) (reviewed in Yen, Semin. Virol. 4:33-42 (1993)) and the herpes simplex virus thymidine kinase (HSV-TK) (McKnight, Nucleic Acids Res. 8:5949-5964 (1980)) genes. Unlike the intronless variants of the highly intron-dependent gene transcripts that usually fail to accumulate in the cytoplasm (Jonsson et al., Nucleic Acids Res. 20:3191-3198 (1992); Nesic et al., Mol. Cell. Biol. 13:3359-3369 (1993); Neuberger & Williams, Nucleic Acids Res. 16:6713-6724 (1988)), these intronless viral transcripts can efficiently accumulate in the cytoplasm without undergoing the process of splicing (Yen, Semin. Virol. 4:33-42 (1993); McKnight, Nucleic Acids Res. 8:5949-5964 (1980); Liu & Mertz, Genes Dev. 9:1766-1780 (1995)). Recent studies have indicated that the cytoplasmic accumulation of unspliced HBV transcripts is facilitated by a specific cis-acting RNA element that interacts with cellular factors (Donello et al., J. Virol. 70:4345-4351 (1996); Huang & Liang, Mol. Cell. Biol. 13:7476-86 (1993); Huang & Yen, Mol. Cell. Biol. 15:3864-3869 (1995)).
In contrast to viral intronless gene expression, much less is known about the expression of cellular intronless genes, which include the genes coding for histone proteins (Kedes, Annu. Rev. Biochem. 48:837-870 (1979)), .beta.-adrenergic receptor (Koilka et al., Nature 329:75-79 (1987)), .alpha.-interferon (Nagata et al., Nature 287:401-408 (1980)) and c-jun (Hattori et al., Proc. Natl. Acad. Sci. USA 85:9148-9152 (1988)). The existence of a cis-acting element in the c-jun message has been suggested (unpublished results, cited in Liu & Mertz, Genes Dev. 9:1766-1780 (1995)).
In many applications, e.g., in research laboratories, transgenic research and gene therapy, genes are expressed from vectors that are designed to make large amounts of the desired mRNA. In a common formulation, a DNA segment obtained by making a complementary copy of a desired mRNA (a cDNA molecule) is inserted into an expression vector downstream of a promoter and just upstream of a polyadenylation signal. Frequently, when this is done in its simplest form, very little expression is observed because much or most gene expression in eukaryotic cells appears to require the splicing of introns in order to allow cytoplasmic accumulation of mRNA. While the reason for this almost general requirement for splicing is unclear, most expression vectors attempt to alleviate this problem by incorporating an intron either upstream or downstream of the coding region contained in the DNA insert. One of the most commonly used introns is the SV40 small t antigen intron. However, this intron can lead to aberrant splicing, and splicing to "cryptic" sites lying within the cDNA of interest (Huang and Gorman, Mol. Cell. Biol. 10:1805-1810 (1990)), resulting in lower than optimal levels of mRNA.